We will test a new model to explain the highly variable rates of genetic adaptation seen in different biological situations. Genetic adaptation is the process by which the genome of a growth-limited population changes by the combined effects of mutation and natural selection. Adaptation underlies how bacteria become resistant to antibiotics, how pathogens evade host defenses and how cancer cells escape growth limiting controls systems in metazoans. In all organisms, mutation types that cause small effects on growth are much more common (1000-fold) than mutations causing large phenotypic changes - gene copy number changes are notably frequent. We propose that stringent selections detect only rare, large-effect mutations while softer selection detect even small-effect mutations which are much more common. Laboratory selections are extremely stringent and have vastly underestimated the rate of mutation formation. Natural selection is generally less severe (softer), but allows for rapid adaptation because it detects the common small-effect mutants. Whenever selection is soft, adaptation improve growth so rapidly that growth limitation appears to be mutagenic, when actually the higher speed reflects detection of a larger fraction of pre-existing mutant types. In testing this model, we use two genetic systems. In both systems, a cell population is plated on medium that prevents growth of the parent and reveals selected mutant colonies that appear over several days, giving the impression that stress of selection is mutagenic. For one system, developed by John Cairns, we can show that the number of mutants appearing under selection is determined prior to selection by the frequency of pre- existing cells with multiple lac copies. Since cells responsible for the colonies arise before selection, they cannot be stress-induced. In the other system, developed by Yang, Lu and Wang, a fully revertant phenotype requires two common mutations. We can show that colonies include some slow-growing cells with one mutation and other faster-growing cells with both mutations - demonstrating the accumulation of mutations during growth under selection. The very first genetic event may be increases in gene copy number, which arise at high frequency before selection. We will test a model for duplication formation, that involves a new mutation type (the sTID = ABCD -> ABCD-D'C'B'A'-ABCD) which forms at a high rate but is usually lost by reversion and counter-selection. Selection for more copies of some gene (e.g. B in the sequence), maintains the sTID and favors cells with secondary deletions that modify the repeat to reduce cost and thereby allow higher amplifications. The modified duplication types are found only after prolonged growth under selection. Copy number variants of these types have been seen in many organisms (including humans) and may be major contributors to genetic adaptation in all systems. PUBLIC HEALTH RELEVANCE: Project narrative We will experimentally test a new view of genetic adaptation that explains why bacterial populations adapt quickly to growth limitation, while human somatic cells adapt very slowly (making cancers rare, given the number of cells at risk). The basis of these ideas is the observation that the most common mutation types cause very small phenotypic effects, which are routinely missed in the laboratory, but can contribute heavily to growth in natural settings. We will extend preliminary evidence that systems cited as evidence for stress-induced mutagenesis are actually situations in which a weak selection allows rapid adaptation by exploiting pre-existing mutants that are common under all growth conditions.